Duchenne muscular dystrophy (DMD) is the most common childhood muscular dystrophy affecting 1/4,000 males worldwide [1]. DMD is caused by mutations in the X-linked DMD gene, which encodes dystrophin, a protein that when absent compromises sarcolemma stability [2-5]. Resulting consequences include a cyclical degeneration and regeneration process in which muscle satellite cells are continuously replacing damaged myofibers [6,7]. Skeletal muscle is further insulted by immune cells that scavenge necrotic tissue, adipose cells replacing dystrophic muscle and fibrosis [8-11]. This culminates in an environment in which skeletal and cardiac muscle is progressively rendered non-functional, leading to respiratory or cardiac complications, and patient death by the third decade of life [12,13]. To date, no FDA approved therapies directly address the underlying genetic defect. Corticosteroids can prolong ambulation for up to 3 years and improve patient quality of life, but are not curative [14,15].
Several potential DMD therapies are in pre-clinical development or ongoing clinical trials [16-19]. The most progressed therapy is antisense oligonucleotide (AO) targeted DMD exon skipping. Two AO chemical backbones, 2-O-methyl (2′OMe) and morpholino (PMO), are in Phase IIb and Phase III clinical trials and target an exonic splice enhancer (ESE) element to skip DMD exon 51, addressing 13% of all DMD mutations [20-24]. After weekly systemic administration in DMD patients AOs rescued dystrophin expression ranging from 0-15% or 47% of normal levels [21,25]. Based on data from transgenic mdx mice and allelic diseases, X-linked dilated cardiomyopathy and Becker muscular dystrophy, it is predicted that 20-30% of normal dystrophin levels may be required for a therapeutic benefit [26-28]. In addition, antisense based therapies have variable exon skipping efficiencies within the same muscle, across muscle types, and between patients and types of deletions indicating potential for improvements [21,23,29].
There are many strategies to address limitations of AO distribution and exon skipping efficacy in vivo [30-33]. The present inventors have focused on finding independent molecular agents that potentiate antisense based exon skipping. Their previous work identified dantrolene, an FDA approved drug that increased AO targeted exon skipping activity in a high-throughput screen (HTS), in human and mouse cell models, and in mdx mice treated with AO provided a functional benefit [33]. Dantrolene and other small molecule inhibitors of the Ryanodine Receptor (RyR1) also increased exon 51 skipping in a patient iDRM, suggesting this as the relevant molecular target. These studies were also published as PCT WO2013/033407, which is incorporated by reference herein in its entirety. This previous work highlights the advantage of a HTS workflow, which identifies compounds with significant biological activity and effectively re-purposes their use. The present inventors have focused in particular on identifying FDA approved drugs that modulated exon skipping activity, given that these drugs typically have known molecular targets and toxicity profiles.
There is a need to identify additional small molecules which potentiate AO exon skipping (e.g. in skeletal muscle), and to identify molecular targets and to better understand relevant pathways and interactions for small molecule potentiation of AO exon skipping in order to identify additional small molecules.